1.1. Technical Field
The present invention relates to a chimeric protein containing an intramolecular chaperone (IMC) like sequence linked to a target protein. In particular, the invention relates to a chimeric protein containing an IMC like sequence linked to an insulin precursor. The present invention also relates to a process for obtaining a correctly folded insulin-precursor-containing chimeric protein, comprising, inter alia, contacting an incorrectly folded chimeric protein containing an IMC like sequence linked to an insulin precursor with at least one chaotropic auxiliary agent. The present invention further relates to an assay for screening an amino acid sequence for the ability to improve folding of an insulin precursor using a chimeric protein containing an IMC like sequence linked to an insulin precursor.
1.2. Background Art
1.2.1. Intramolecular Chaperones and Protein Folding
Molecular chaperones are defined as a class of proteins that assist correct folding of other polypeptides but are not components of the functional assembled structure (Shinde and Inouye, TIBS, 1993, 18:442-446). Intramolecular chaperones (IMCs) are part of the precursors of the target proteins to be folded and in their absence the target protein molecules do not have enough information for proper self-folding (Inouye, Enzyme, 1991 45:314-321). Unique features of IMCs include: a) the IMC and the target protein are linked by a peptidyl bond forming a single polypeptide; b) the IMC is absolutely required for the formation of active conformation of the target protein, but not required for the function of the target protein; c) upon completion of the protein folding, the IMC is removed either by autoprocessing or by another endopeptidase; d) the IMC does not function as a catalyst, i.e., one IMC molecule is able to refold only one molecule of the target protein; and e) the IMC is a highly specific “tailor-made” polypeptide which works only for the target proteins (Inouye, Enzyme, 1991, 45:314-321).
Recently, it has been shown that an IMC or propeptide can help the target protein fold intermolecularly, i.e., the IMC or propeptide is not linked to the target protein via a peptidyl bond, but rather is added to the folding reaction as a separate peptide (U.S. Pat. No. 5,719,021). However, it is noteworthy that the propeptide used in the U.S. Pat. No. 5,719,021 is the natural propeptide of the target protein or a propeptide of a polypeptide that has the same function of the target protein and the polypeptide also has an amino acid sequence that is similar to the target protein. In addition, the intermolecular reaction described in the U.S. Pat. No. 5,719,021 must be carried out in a buffered ionic aqueous medium favoring hydrophobic interaction.
Examples of IMCs include the propeptides of subtilisin, α-lytic protease, carboxypeptidase Y and ubiquitin (Shinde and Inouye, TIBS, 1993, 18:442446). Certain characteristics of an IMC sequence of subtilisin include: a) the IMC contains a higher percentage of charged amino acid residues than the target protein; b) the distribution of these charged residues within the IMC is extremely uneven, i.e., the N-terminal half contains more positively charged residues than negatively charged residues and the C-terminal half contains more negatively charged residues than positively charged residues; c) Ser and Thr residues within the IMC are also unevenly distributed; d) the IMC contains a reactively high content of aromatic residues, and e) the IMC contains a hydrophobic sequence of 9 residues (Inouye, Enzyme, 1991, 45:314-321). A similar bias towards charged residues is also observed in α-lytic protease and carboxypeptidase Y (Inouye, Enzyme, 1991, 45:314-321).
1.2.2. Amino Acid Sequence of Mature Human Growth Hormone
The amino acid sequence of mature human growth hormone (hGH) is disclosed in Ikehara et al., Proc. Natl. Acad. Sci. USA, 1984, 81:5956-5960. There is no suggestion in the art that mature hGH or any portion thereof can function as an IMC or propeptide. Actually, mature hGH or any portion thereof can not be considered a propeptide at all because by definition, any pre, pro, or prepro sequence is removed from a mature sequence.
1.2.3. Human Insulin Structure
Insulin is a well-defined peptide with known amino acid sequence and structural characteristics (Watson et al., Recombinant DNA—A Short Course; Scientific American Books, W. H. Freeman Co., New York, 1983, pp. 231-235; Norman and Litwack, In Hormones, Academic Press, New York, 1987, pp. 264-317). This hormone consists of two separate peptide chains which are the A chain (21 amino acids) and the B chain (30 amino acids) joined by disulfide bridges as indicated in FIG. 1B. Proinsulin is the biological precursor of insulin and is a single peptide chain formed when the A and B chains are connected by the C peptide (FIG. 1A).
1.2.4. Human Insulin Produced by Recombinant Methods
Human insulin was the first animal protein made in bacteria in a sequence identical to that of the human pancreatic peptide (Watson et al., Recombinant DNA—A Short Course: Scientific American Books, W. H. Freeman Co., New York, 1983, pp. 231-235). The first successful expression of human insulin in laboratory was announced in 1978 and human insulin was approved as a therapeutic drug in 1982 (Johnson, Science, 1983, 219:632-637).
1.2.4.1. Two-Chain Method
According to this method, each insulin chain is produced as a β-galactosidase (β-gal) fusion protein in separate fermentations using E. Coli transformed with plasmids containing a DNA sequence encoding the A or B chain of human insulin, respectively. The products are intracellular and appeared in prominent cytoplasmic inclusion bodies (Williams et al., Science, 1982, 215(5):687-689). Recombinant proteins produced in E. Coli usually represent 10-40% of the total protein (Burgess, Protein Engineering; Oxender, D. L., Fox, C. F., Eds.; Alan R. Liss, Inc.; New York, 1987; pp. 71-82.).
Once removed from the inclusion bodies, chemical cleavage by CNBr at the Met residue between the β-galactosidase and the A or B chain, followed by purification, gave separate A and B peptides. The peptides are then combined and induced to fold at a ratio of 2:1 of A-B chain (S-sulfonated forms) in the presence of limited amounts of mercaptan in order to obtain an active hormone (Chance et al., In Peptides: Synthesis-Structure-Function, Rich D. M. Gross, E., Eds., Pierce Chemical Co., Rockford, Ill. 1981, pp.721-728, Frank and Chance, In Quo Vadis? Therapeutic Agents Produced by Genetic Engineering, Joyesuk et al., Eds., Sanoff Group, Toulouse-Labege, France, 1985, pp. 137-148). After 24 h, the yield is approximately 60% based on the amount of B chain used (Chance et al. In Insulins, Growth Hormone and Recombinant DNA Technology, Raven Press, New York. 1981, pp. 71-85; Johnson, Fluid Phase Equilib., 1986, 29: 109-123). Goeddel et al., Proc. Natl. Acad. Sci. U.S.A., 1979, 76(1):106-110, obtained similar results with 20% of the total cellular protein expressed as either the A or B chain fusion protein. Subsequent folding of S-sulfonated chains give 50-80% correct folding.
The large size of the β-gal fusion protein limits yields since the fusion protein of β-gal (−1000 amino acids) and insulin A or B chain (21 or 30 amino acids, respectively) became detached from the cell's ribosome (premature chain termination during translation and therefore yields incomplete insulin peptides (Burnett, Experimental Manipulation of Gene Expression, Inouye, Ed., Academic Press, New York, 1983, pp. 259-277; Hall. Invisible Frontiers—The Race to Synthesize a Human Gene, Atlantic Monthly Press, New York, 1987). A key improvement to this approach is the use of the tryptophan (Trp) operon in place of the lac operon (β-gal system) to obtain a smaller fusion protein. The Trp operon consists of a series of five bacterial genes which sequentially synthesize the enzymes responsible for the anabolism of tryptophan. One of these enzymes, Trp E, has only 190 amino acids compared to β-gal's 1000 amino acids. The Trp E gene followed by genes for the A or B chains of insulin has the added advantage of enhancing fusion protein production from 5-10% to 20-30% of the total protein (Hall, Invisible Frontiers—The Race to Synthesize a Human Gene, Atlantic Monthly Press, New York, 1987) since the Trp promoter is a strong promoter in E. Coli. This leads to at last 10-fold greater expression of polypeptide when compared to the lac (i.e., β-gal) system (Burnett, Experimental Manipulation of Gene Expression, Inouye, Ed., Academic Press, New York, 1983, pp. 259-277). The Trp operon is turned on when the E. Coli fermentation runs out of tryptophan (Hall, Invisible Frontiers—The Race to Synthesize a Human Gene, Atlantic Monthly Press, New York. 1987; Etienne-Decent, In Genetic Biochemistry: From Gene to Protein, Ellis Horwood Limited, Chichester, U.K., 1988, pp. 125-127). This characteristic is beneficial during fermentation since cell mass can first be maximized. Then, when appropriate, the cell's insulin production system can be turned on by allowing the fermentation media to become depleted in Trp.
After fermentation is completed, the cells are recovered and disrupted. The cell debris is than separated from the inclusion bodies, and the inclusion bodies are dissolved in a solvent, although specifics are not known (Wheelwright, Protein Purification, Oxford University Press; New York, 1991, p. 217). Inclusion bodies are sometimes dissolved in 6 M guanidine HCl and 0.1 mM dithiothreitol (Burgess, Protein Engineering, Oxender and Fox, Eds., Alan R. Liss, Inc., New York, 1987, pp. 71-82). Next, the Trp-LE-Met-A chain and the Trp-LE-Met-B chain undergo a CNBr cleavage to release the A and B insulin chains. Further modifications of the A and B chains include oxidative sulfitolysis, purification and combination to produce crude insulin. This crude insulin is subjected to ion exchange, size exclusion, and reversed-phase high-performance liquid chromatography (RP HPLC) to produce the purified recombinant human insulin (Frank and Chance, Munch Med. Wschr. 1983, 125(Suppl. 1):514-520).
1.2.4.2. Proinsulin Method (Intracellular)
Human insulin can also be made with recombinant microorganisms that produce intact proinsulin instead of the A or B chains separately (Kroeff et al., J. Chromatogr, 1989, 481:45-61). Initially, mRNA is copied into cDNA, and a methionine codon is chemically synthesized and attached to the 5′ end of the proinsulin cDNA. The cDNA is inserted into a bacterial gene in a plasmid vector that is introduced and then grown in E. Coli. Proinsulin can be released from the bacterial enzyme (β-gal) fragment (or alternatively from the Trp-LE/Met Proinsulin (Trp proinsulin) by destroying the methionine linker. The proinsulin chain is subjected to a folding process to form the correct intramolecular disulfide bridges, and the C peptide can then be cleaved with enzymes to yield human insulin (Frank and Chance, Munch Med. Wschr., 1983, 125(Suppl. 1):514-520). In comparison, the two-chain method previously described is more complex.
Dorschug et al. constructed recombinant plasmid encoding fusion proteins containing a mini-proinsulin (B-Arg-A), expressed the fusion proteins in E.coli (inclusion body) and yeast (secreted), prepared correctly folded mini-proinsulin via BrCN cleavage and oxidative sulfitolysis, and converted the correctly folded mini-proinsulin into human insulin by treatment with trypsin and carboxypeptidase B (EP 0,347,781 B1; IL 9,562,511 B and AU 611,303 B2).
Tottrup and Carlsen, Biotechnol. Bioeng, 1990, 35:339-348 used the yeast system in an optimized batch-fed fermentation, yields of the fusion protein of superoxide dismutase-human proinsulin (SOD-PI) were reported to be 1500 mg/L. SOD-PI would be the starting material for the production of recombinant human insulin; yields of the final product have not been reported.
Recently, Castellanos-Serra et al., FEBS Letters, 1996, 378:171-176 expressed in E. Coli a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide (1-22 amino acid residues) linked to the N-terminus of proinsulin by a lysine residue. The chimeric proinsulin was isolated from inclusion bodies, refolded via oxidative sulfitolysis, and then converted into the correctly fusion proteins insulin by prolonged reaction with trypsin and carboxypeptidase B. The IL2-proinsulin fusion can be folded correctly without first removing the IL2 fragment, thus eliminating the cyanogen bromide and the associated purification steps. However, the step of oxidative sulfitolysis and the associated purification steps cannot be avoided by the use of IL2-proinsulin fusion protein.
1.2.4.3. Proinsulin Method (Secreted)
Villa-Komaroff et al., Proc. Natl. Acad. Sci. U.S., 1978, 75(8):3727-3731 were first to describe a secretion system for human proinsulin in E. Coli. Thim et al. constructed recombinant plasmids encoding fusion proteins containing a modified yeast mating factor α I leader sequence and an insulin precursor (Thim et al., Proc. Natl. Acad. Sci. USA, 1986, 83:6766-6770). The leader sequence serves to direct the fusion protein into the secretory pathway of the yeast cell and to expose the fusion protein to the Lys-Arg processing enzyme system. Partial processing also occurred at one or both dibasic sequences between B and A chains within proinsulin and other insulin precursors containing a short spacer peptide (containing 6 or more amino acid residues) in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g. B-Arg-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors could enzymatically be converted into insulin by treatment with trypsin and carboxypeptidase B Diers et al., Drug Biotechnology Regulations (Scientific Basis and Practices), Chiu and Gueriguian, Eds., Marcel Dekker, Inc., New York, 1991, pp. 167-177, describe the unfolded peptide as a leader or prosegment, next a Lys-Arg sequence, the B chain (amino acids 1-29), a short peptide bridge, followed by the A chain (amino acids 1-21). In this precursor, amino acid 29 of the B chain of insulin is connected to amino acid 1 of the A chain by a short connecting peptide containing one basic amino acid adjacent to the A chain. Human insulin is produced through transpeptidation followed by hydrolysis of the ester bond formed. Several chromatography steps follow for further purification.
1.2.5. Folding of Insulin Precursors
Human insulin is a protein possessing two amino acid chains of 51 amino acid residues in all. Six cysteine residues are present in the two amino acid chains, which in each case two cysteine residues being linked to each via a disulfide bond. Statistically, there are 15 possibilities of forming disulfide bridges within one human insulin molecule. However, only one of the 15 possibilities exists in biologically active human insulin with the following disulfide bridges: 1) A6-A11; 2) A7-B7; and 3) A20-B19.
The formation of the disulfide bridges which are present in human insulin is effected by way of an intermediate, with the cysteine residues of the human insulin being provided with a sulfur protective group, e.g., with a S-sulfonate (—S—SO3−) group (EP 0,037,255). In addition, pig proinsulin in which the cysteine residues are present as thio residues (—SH) has been used to obtain proinsulin possessing correctly linked cysteine bridges (Biochemistry, 1968, 60:622-629). Obermeier et al. described a process for obtaining proinsulin possessing correctly linked cysteine bridges from a corresponding proinsulin amino acid chain at a concentration of 0.05 to 0.3 g per liter in the presence of mercaptan, chaotropic auxiliary agents and hydrophobic absorber resins (U.S. Pat. No. 5,473,049). The step of oxidative sulfitolysis is eliminated in the process described in U.S. Pat. No. 5,473,049. However, the insulin protein can only be folded at a low concentration, which greatly diminishes the commercial value of this process. In addition, the use of large amount of mercaptan and hydrophobic absorber resins increase process complexity and down-stream purification costs. From the disclosure of the U.S. Pat. No. 5,473,049, it is unclear whether the benefit of eliminating the step of oxidative sulfitolysis step will outweigh the increased down-stream purification costs.
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.